First detection and characterization of mcr-1 colistin resistant E. coli from wild rat in Bangladesh

Colistin resistance is a global concern warning for a one health approach to combat the challenge. Colistin resistant E. coli and their resistance determinants are widely distributed in the environment, and rats could be a potential source of these isolates and resistant determinants to a diverse environmental setting. This study was aimed to determine the presence of colistin resistant E. coli (CREC) in wild rats, their antimicrobial resistance (AMR) phenotypes, and genotypic analysis of mcr-1 CREC through whole genome sequencing (WGS). A total of 39 rats were examined and CREC was isolated from their fecal pellets onto MacConkey agar containing colistin sulfate (1 μg/ mL). AMR of the CREC was determined by disc diffusion and broth microdilution was employed to determine MIC to colistin sulfate. CREC were screened for mcr genes (mcr-1 to mcr-8) and phylogenetic grouping by PCR. Finally, WGS of one mcr-1 CREC was performed to explore its genetic characteristics especially resistomes and virulence determinants. 43.59% of the rats carried CREC with one (2.56%) of them carrying CREC with mcr-1 gene among the mcr genes examined. Examination of seventeen (17) isolates from the CREC positive rats (n = 17) revealed that majority of them belonging to the pathogenic phylogroup D (52.94%) and B2 (11.76%). 58.82% of the CREC were MDR on disc diffusion test. Shockingly, the mcr-1 CREC showed phenotypic resistance to 16 antimicrobials of 8 different classes and carried the ARGs in its genome. The mcr-1 gene was located on a 60 kb IncI2 plasmid. On the other hand, ARGs related to aminoglycosides, phenicols, sulfonamides, tetracyclines and trimethoprims were located on a 288 kb mega-plasmid separately. The mcr-1 CREC carried 58 virulence genes including genes related to adhesion, colonization, biofilm formation, hemolysis and immune-evasion. The isolate belonged to ST224 and closely related to E. coli from different sources including UPEC clinical isolates from human based on cgMLST analysis. The current research indicates that rats might be a possible source of CREC, and the presence of mcr-1 and other ARGs on plasmid increases the risk of ARGs spreading and endangering human health and other environmental components through this infamous pest.


Introduction
Colistin is a reserve group of antibiotics.Colistin resistance became one of the key concerns due to its importance in treating MDR infections particularly with β-lactam and carbapenem resistance [1].Colistin resistance is increasingly reported in the members of Enterobacteriaceae especially in E. coli.Although E. coli is a normal flora of the gastrointestinal tract of all warm-blooded animals, certain strain has become pathogenic through the acquisition of virulence genes or resistance through horizontal transfer and are associated with a range of intestinal and extra-intestinal infections [2,3].
Colistin resistance is mediated by intrinsic adaptation, mutation or horizontal acquisition of colistin resistance [4].Mobile colistin resistance (mediated by mcr genes) is a pivotal concern among the colistin resistance mechanisms due to rapid dissemination among different bacterial host through mobile genetic elements like plasmid and transposons [4,5].So far ten mcr genes (mcr-1 to mcr-10) have been reported since the discovery of the first colistin resistant gene, mcr-1 remained as the predominant one [6,7].Colistin resistant E. coli is widely distributed in the environment.E. coli carrying mcr genes was reported from different sources including human, street food, surface water, urban sludge, poultry meat and feces, and poultry farm environment [8][9][10][11][12][13][14].However, no study has yet described the occurrence of colistin resistant especially mcr-1 E. coli from rodents such as rat in Bangladesh.
Rodents are reservoirs or carriers of MDR pathogens [15][16][17][18][19]. From one health perspective, rodents play important role in disseminating pathogens into the ecosystem to affect other hosts including wildlife, domestic animals and humans.Because of their inherent nature of dwelling human and animal food resources, rats are frequently seen in the household and farm environment especially poultry farms in Bangladesh.Recently, colistin resistant and MDR E. coli has been reported from fecal samples and poultry farm environments in Bangladesh and other part of the world [10,14,20] indicating a high chance of rats being exposed to such critical pathogens.And because of shared habitat, rats might be involved in the transmission of AMR including colistin resistant E. coli to human and livestock especially to poultry farms.Thus, the present study was conducted to examine the occurrence of colistin resistant E. coli (CREC), determination of mcr genotypes and characterization of a mcr-1 E. coli isolated from rats dwelling animal rearing facility through whole genome sequencing (WGS) based approach.

Ethical approval
This study design was approved by the Animal Welfare and Experimentation Ethics Committee, Bangladesh Agricultural University [Approval no.AWEEC/BAU/2021 (48)].

Sample collection
A total of thirty-nine (39) wild rats were caught from the animal rearing facility (24.72544N90.43806E)during the period from October 2021 to June 2022.Fecal pellets were directly collected from the rat intestine after sacrificing by cervical dislocation.Three to four fecal pellets were collected from the distal part of the rat intestine in sterile Eppendorf tube and processed immediately for the isolation of CREC.

Isolation of CREC
Fecal pellets were suspended in 1 mL Phosphate Buffer Saline (PBS) by repeated vortexing.Serial 10-fold dilution was prepared from the fecal suspension in PBS and 100 μL of the suspension (non-diluted, 10-fold, 100-fold diluted) was spread onto MacConkey agar medium containing colistin sulfate 1 μg/mL (Fujifilm Wako Chemical Corporation, Osaka, Japan) [9].After overnight (~16 h) incubation at 37˚C, E. coli specific lactose-fermenting colonies were streaked onto fresh MacConkey agar plates containing colistin sulfate (1 μg/mL) to obtain a pure culture.Genomic DNA was extracted from the E. coli suspected isolates by boiling method followed by PCR targeting the malB gene [21] using the primers furnished in S1 Table .Colistin resistance in the isolated E. coli was further confirmed by culturing the colonies in Luria Bertani (LB) broth containing colistin sulfate @ 2 μg/mL and stored at -20˚C for further uses.

Detection and subtyping of mobile colistin resistance (mcr) genes
CREC isolated in this study was screened for mcr genes (mcr-1 to mcr-10) by PCR.For mcr-1 to mcr-5 a multiplex PCR was employed [22].The multiplex PCR was conducted in a final 20 μL reaction containing 10 μL one Taq 1 Quick-Load 1 2X master mix (New England Biolabs Inc., Ipswich, MA, USA) and 5 pmol of each primer (S1 Table ).E. coli strain 1ChBEc2mcr (mcr-1 positive) [9] and E. coli strain ATCC25922 was used as the positive and negative controls, respectively.On the other hand uniplex PCR was performed for the detection of mcr-6 to mcr-10 following the in-house protocol developed by Awasthi et al.

Phylogenetic grouping of E. coli
CREC isolated in this study were examined for their phylogenetic group by PCR targeting chuA, yjaA, and TspE4.C2 DNA fragments [24].PCR was performed in a final 20 μL volume with One Taq 1 Quick-Load 1 2X master mix (Biolabs) and 10 pmol of each primer followed by amplification at the conditions mentioned in S1 Table.

Antibiotic susceptibility testing and minimum inhibitory concentration (MIC) determination
Disc diffusion test was performed to determine the antibiotic susceptibility of CREC isolated in this study [25].A total of 21 antibiotics of 10 classes regularly prescribed in animal production and human clinical cases in Bangladesh, were tested (S2 Table ).Each antibiotic was assessed at least three times to confirm the reproducibility of the results and E. coli strain ATCC25922 was used as the control strain.Isolates showing resistance to three or more classes of antibiotics were considered as MDR [26].The MIC of colistin sulfate was determined by the broth microdilution method [25].

Whole genome sequencing (WGS) and analysis
To explore the genomic characteristics and location of mcr-1 gene, the mcr-1 CREC strain isolated in this study (strain RJWEcMCR-1-BAU) was subjected to WGS on an Illumina Nextseq 550 platform (Illumina, CA, USA) commercially.The quality of FASTQ sequences and trimming of the low-quality reads were performed on Trimmomatic (Galaxy Version 0.38).Assembly of the trimmed reads were performed on a hybrid assembler Unicycler (Galaxy Version 0.4.8.0) followed by annotation through NCBI Prokaryotic Genomes Annotation Pipeline (PGAP).In addition, to explore the functional features RAST (Rapid Annotation using Subsystem Technology) server [27] was used.
The comparative ring image of the RJWEcMCR-1-BAU genome was prepared on BRIG (BRIG dist 0.95, [28]) and BacWGSTdb 2.0 server [29] was explored for the prediction of sequence types, core genome MLST analysis (cgMLST) and source tracking of the genome.Detection of ARGs and VFGs were performed on the Comprehensive Antibiotic Resistance Database (CARD) [30], and VirulenceFinder [31] databases, respectively.The E. coli 1ChBEc2mcr genome was annotated against the PlasmidFinder [32] to detect plasmid replicons and the plasmid containing mcr-1 gene was revealed through reference mapping on Geneious prime platform version 2022.1.1 (Biomatters).In addition, Proksee tools (Prokka, CARD Resistance Gene Identifier, Phigaro, mobileOG-db and CRISPR/Cas Finder [33] and Mobile Element Finder (MEF) version v1.0.3 (2020-10-09)) [34] were used to generate the circular images with distribution of contigs, CDS, ARGs and CRISPAR/Cas clusters, and mobile genetic elements (MGE) in the study genome.For mobile element detection 90% minimum coverage with 90% sequence identity and 30 maximum truncation filters were used.

Occurrence of colistin resistant mcr-1 E. coli (mcr-1 CREC) in rat
Selective culture in presence of colistin sulfate followed by PCR targeting the malB gene revealed 17 rat feces positive for CREC.PCR targeting mcr genes (mcr-1 to mcr-10) revealed 1 rat carrying mcr-1 and the others (16/17) carried non-mcr CREC according to the screening method.Overall, the occurrence of CREC and mcr-1 CREC in the rats was recorded as 43.59% and 2.56%, respectively.

Phylogenetic grouping of CREC
Most of the CREC isolated in this study belonged to pathogenic phylogroup of E. coli (64.70%) with 52.95% in the pathogenic phylogroup D and 11.76% in the group B2.On the other hand, 35.29% of the isolates belonged to commensal E. coli of phylogroup B1 (Table 1).

General characteristic of the RJWEcMCR-1-BAU genome
The genome was assembled to 126 contigs with an estimated size of 5203305 bp with 50.5% GC contents which corresponds to a standard E. coli genome.The longest and shortest contig size was 447963 and 268 bp, respectively with a mean length of 41296.1 bp (Table 3).The annotated genome consisted of 5039 coding sequences and 81 RNAs (rRNAs: 7, tRNAs: 65 and ncRNAs: 9).The genome comprises of eight ( 8) CRISPR regions and one Cas cluster with

Genetic relatedness of RJWEcMCR-1-BAU
The genome RJWEcMCR-1-BAU belongs to ST224 based on whole genome based cgMLST analysis and is closely related to E. coli reported from different parts of the world (Fig 1).The closest neighbor of this isolate is an E. coli (strain C103) reported from urine of a human in France which differ by 61 alleles.The isolate was also found very close to E. coli reported from chicken, pig, cow, dog and horses indicating its potential to interspecies dissemination.The RJWEcMCR-1-BAU genome was compared with E. coli strain Sakai (Accession no.GCA_000008865.2), the closest neighbor (E. coli strain C103, accession no.GCA_013047065.1),E. coli isolated from chicken meat in Bangladesh (strain 1ChBEc2mcr, accession no.GCA_027757765.1) and the sequences were found highly conserved; however, a significant number of non-homologous sequences were also evident throughout the genomes (Fig 2 ) which might be linked to the jumping or transposable sequences.

Location of mcr-1 and other plasmid mediated ARGs the RJWEcMCR-1-BAU genome
Raw reads of the RJWEcMCR-1-BAU genome were mapped with mcr-1 gene carrying plasmids reported earlier (S3

Discussion
Rats are cosmopolitan and well adapted to diverse ecosystems.They are frequently encountered in human habitats and animal rearing facilities.Due to exposure to multiple environmental settings, they come in close contact with potential pathogens, and might become a potential source of pathogens and/ or resistant determinants especially AMR to humans and animals [17,35,36].The occurrence of bacterial pathogens in rats caught from animal rearing facilities including poultry and pig farms has earlier been reported [37].However, occurrences of bacterial pathogens, especially AMR in Rats remained untouched in Bangladesh.Recent reports on the occurrence of AMR in poultry farm environments [10,14], urban sludges [11] and abundance of rats in the animal rearing facilities, prompted us to initiate this work.Colistin resistant E. coli are major public health concern.In this study we focused on the occurrence of colistin resistant E. coli in rat considering the increased trend of colistin resistance and potential of rats to transmit this notorious pathogen to humans or animals.This study encountered colistin resistant E. coli in wild brown rats dwelling animal rearing facility and 64.7% of the isolates belonged to pathogenic phylogroup.Occurrence of colistin resistance in rat E. coli has been reported earlier [36] but to the best of our knowledge this is the first such report from Bangladesh.Remarkably, around 58.82% of the CREC are MDR and resistant to antimicrobials commonly used from human and animal practices in Bangladesh.Occurrence of MDR in CREC from rats is very alarming as they might act as the potential source of this resistant bacterium to their counterparts.
Colistin resistance is mediated by several mechanisms encoded by chromosomally mediated genes resembling to that of natural colistin resistant bacterium, and members of the mcr gene family present on the horizontally transferable elements like plasmids and transposons [4,5].In this study, only one of the CREC carried mcr-1 and the others were non-mcr-1-10.indicating that the non-mcr-1-10 isolates might carry chromosomally mediated antimicrobial resistance as reported earlier from different part of the world including Bangladesh [10,38].To determine whether the mcr-1 positive strain harbors chromosomal mutations crucial for colistin resistance, we scrutinized the amino acid sequences of PmrCAB, PhoPQ, PmrD, and MgrB, as outlined previously [39].While no mutations were detected in PmrA, PmrC, PhoP, PhoQ, and MgrB, this strain exhibited mutations at D283G and Y358N in the PmrB, and K82T in the PmrD (S3 Data).Although these mutations have been identified in colistin-resistant E. coli strains, their experimental validation remains lacking [38,40,41].However, the potential contribution of these mutations to the colistin resistance observed in our study strain cannot be disregarded.Hence, we recommend further studies to establish the association of these chromosomal mutation with the colistin resistance of E. coli.Interestingly, mcr-1 carrying CREC had an elevated MIC (� 8 μg/ mL) than the other CREC (� 2 μg/ mL) which corresponds with the results from previous studies who described an elevated MIC in mcr-1 E. coli than non-mcr E. coli [10,41].Supporting the findings that production of mcr-1 in E. coli leads to 4-8 fold increase in the MIC [42], the later CREC might belong to the non-mcr E. coli.
Mobile colistin resistance gene mcr-1 has been reported on horizontally transferable plasmids as well on the chromosome flanked by a transposable element such as ISApI1 [4,5,9,43].In this study, the mcr-1 gene was found to be located on a highly transferable IncI2 like plasmid as reported earlier [4]; however, no ISApI1 or similar transposon but nikB and mobC transfer related genes were located upstream of the mcr-1 gene.Although the role of nikB and mobC in mcr-1 gene transfer is not understood bacterial loss of ISApI1 have been published earlier [44] and speculated to be a part of bacterial adaptation to the environmental changes and essential for the maintenance of mcr-1 gene [45].Previously occurrence of mcr-1 gene on IncF, IncHI2 plasmids was reported in Bangladesh [9,46] but the occurrence of this in IncI2 plasmid is the first report incorporating additional information on the diversity of plasmids carrying mcr-1 genes in this country.
In addition to resistance to colistin, bacterium RJWEcMCR-1-BAU showed resistance to 16 antimicrobials of 8 different classes.ARGs identification through CARD revealed the presence of all the ARGs related to the phenotypic resistance of this bacterium.Moreover, this bacterium carried ARGs conferring resistance to 15 different antimicrobial classes.Interestingly, blast homology search of the genome sequence indicated that most of the ARGs were associated with plasmid-like sequences.Although the location of all ARGs couldn't be confirmed due to short length sequencing, this study identified a 288 kb megaplasmid of highly transferable IncHI2 family that carries ARGs conferring resistance to aminoglycosides {aadA2, ant (3")-IIa, aph(3')-IIa, aph(3")-Ib, aph(6)-Id}, phenicols (cmlA1), sulfonamides (sul1, sul3), tetracyclines (tetA, tetR) and trimethoprims (dfrA15).Occurrence of MDR with mcr-1 E. coli has been reported earlier [9,47], however, rats carrying these types of resistance determinants on plasmids are firstly reported in this study to the best of our knowledge.Occurrence of MDR E. coli carrying ARGs on horizontally transferable elements is worrisome and imposes a potential threat of transferring and disseminating these resistance determinants to human and animals through contaminated food, water and environment.
Source tracking through BacWGStdb and cgMLST revealed the isolate RJWEcMCR-1-BAU belonged to ST224 and closely related to E. coli isolated from humans and animals.Strikingly, the closest neighbor was a clinical strain of E. coli isolated from human urine from France (Accession no.NZ_JABBFG010000000) which differs from the study strain by only 61 alleles.More importantly, the study strain possesses virulence genes related to adhesion, colonization, biofilm formation, enterobactin biosynthesis and iron acquisition, hemolysis and dodging host immunity which are important virulence determinants in ExPEC especially UPEC [48], indicating the strain RJWEcMCR-1-BAU might be a UPEC; however, their UPEC potential need to be further investigated in detailed through phenotypic analysis.
The current investigation concentrated on the presence of CREC and the genomic characterization of a mcr-1 CREC, isolated from a limited number of samples.Due to fund limitation, our primary goal was not to ascertain the prevalence of such CREC in rats; hence, we did not perform sample size calculations and screened a relatively small number of samples.Furthermore, the rats involved in this research were obtained from a singular source, limiting the generalizability of the study's outcomes.Consequently, we recommend further studies with a larger sample size, considering spatiotemporal variations, and encompassing a diverse array of rat species to bolster the robustness and applicability of our findings.

Conclusion
Colistin-resistant E. coli (CREC) is major health concern.This is the first study in Bangladesh describing the occurrence of colistin-resistant E. coli (CREC) and the presence of mcr-1 and other antimicrobial resistance genes on plasmids in CREC isolated from rats.The identification of MDR CREC in rats raises concerns about the potential dissemination of these pathogens to various components of the one health system they interact with.However, further investigations with an expanded sample size, encompassing diverse locations and rat species from various habitats, are necessary to better understand and mitigate the threat of transferring these pathogens and/or resistance determinants to different environmental components, including humans and animals.